Actin is one of the most abundant human proteins, but very little understanding exists about why actin is so highly conserved. Over 350 million years of evolution there have been no amino acid changes in the skeletal striated muscle isoform of actin. Similarly, there is no understanding of why most sequence differences are between tissue-specific isoforms and not between species. The hypothesis that is being investigated in this research is that many allosteric linkages exist within the actin filament, placing constraints and selective pressure on all of the buried residues. These allosteric linkages are essential for the many functions of actin, from muscle contraction to cell motility to forming the stereocilia responsible for hearing, and we postulate that mutations of buried residues that break these allosteric linkages are responsible for a number of diseases. Past productivity has been very strong in this project, but the proposed period represents a major transformation. With our Titan Krios and Falcon II direct electron detector we can have near-atomic resolution for the interactions within the actin filament and between actin and actin-binding proteins. The eight proposed aims include studies of actin mutants, and many complexes of actin filaments with other proteins, including the nucleocapsid (NC) domain of HIV-1. Some of these actin-binding proteins contain structurally conserved domains, such as the Immunoglobulin (Ig) domains, and the hypothesis is that the presence of such domains tells us only about the ancestry and evolution of these proteins and not about how these domains interact with actin. Thus, different Ig domains may interact in a completely different manner with actin, or not interact at all, as was shown for the CH domain within the eponymous protein calponin. The proposed studies will be done in collaboration with other laboratories using biochemistry and mutagenesis, and the resulting work should have a large impact on our understanding of how actin functions in a huge number of cellular processes and interacts with many other proteins.